Today it remains difficult to measure the absorbance of samples at high spatial resolution, despite the importance of the infrared, IR (or visible and ultraviolet, UV) absorbance for chemical analysis.
In the far-field, one generally chooses to achieve high resolution chemical imaging by using the shorter wavelength in the visible or near-IR spectrum, typically up to 1 μm wavelength. Indeed the shorter the wavelength the better the resolution, according to the Abbe criterion. One can also use several approaches such as CARS (coherent anti-Stokes spectroscopy) or SRS (stimulated Raman spectroscopy) to obtain chemical imaging. These probe characteristic Raman signature of a sample. A schema for sub-diffraction CARS microscopy has been described in US 2010/0238438 A1 that uses a single pump beam overlapped in time and space into the sample with two interfering ‘Stokes’ beams of different spatial profile.
IR absorption provides complementary information to Raman spectroscopy and is typically paired with Raman. The obvious advantage of IR absorption is that the cross-section of IR absorption is larger than that of Raman scattering, affording, in principle, a higher sensitivity. The best IR absorption imaging (or IR micro-spectroscopy) is currently carried out using synchrotron IR sources and only affords a diffraction limited resolution (at least several μm).
There is currently no demonstrated far-field methods to improve the resolution of IR absorption imaging up to (or better than) the level of CARS or SRS. To achieve high-spatial resolution when measuring the IR absorbance, one currently needs to exploit near-field probing methods. These are realized by scanning a nanoscale probe in the vicinity of the sample. Resolutions down to several tens of nm (but typically 100-150 nm) have been demonstrated. By nature these techniques are suited to probe the surface of samples, which limit the field of applications. Other publications in the field include patent publication number GB2477817. Furthermore there are inherent difficulties in obtaining probes that effectively work.
Current far-field sub-diffraction microscopies can use the RESOLFT (REversible Saturable OpticaL Fluorescence Transitions) and localization methods (e.g., STORM, PALM, etc). These methods measure the fluorescence of fluorescent molecules that are (in all except very few specific cases) added to samples. These fluorescent molecules are known as chromophores or more generally as ‘labels’. DE4416558 and US 2011/0215258 A1 describe implementation of RESOLFT approach. They are thus not suitable for label-free chemical imaging.
Saturated structured-illumination microscopy (SSIM) has been proposed by Gustafson (M. G. L. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 2005, Vol. 102, 13081-13086). However SSIM is also realized in the case of fluorescent molecule and is not designed to record the IR absorbance (nor the visible/UV one). SSIM uses a complex image processing and is a wide-field approach. A similar schema using a selectively directed, focused illumination beam and a wide field array detector has been proposed in PCT patent publication number WO2010/101894 A2.
Moreover, a paper published by Wilson et al (T. Wilson and D. K. Hamilton, Optica Acta 1984, Vol. 31, 453-465) describes a schema where a single point source of light, in its virgin state i.e. without any modification, goes on a sample that is being scanned for imaging. The resultant beam from the sample is then split into two beams and the differential signal is established as the difference in intensity measured by two different detectors, a confocal detector and a large area detector.
US2012/0097865 describes a differential scheme of improving resolution of visible imaging based on luminescence (fluorescence and similar effects) and requires the addition of ‘labels’. In this technique the luminescence emitted from the labels in the sample is measured at sub-diffraction resolution by using two beams, partially but not completely overlapping on the sample, to excite the luminescence. Other publications in the field include patent publication numbers WO2007106069 and US2006119934.
It is an object of the invention to provide a method and system based on a measure of the absorption of light (IR, visible or UV) by a sample for its sub-diffraction imaging in the far-field.